Fig. 5: GCase activity and level is altered in HEK293T cells in which LRRK2 kinase activity is pharmacologically or genetically manipulated.

a Representative western blot of LRRK2-Flag, pSer935 LRRK2 and GCase in HEK293 cells overexpressing empty vector (EV), LRRK2 WT and LRRK2 G2019S and treated with the LRRK2 inhibitor MLi2 100 nM for 90’. b From left to right, quantification of the GCase activity (normalized by total proteins), GCase levels normalized by β-actin and GCase activity normalized by GCase level from western blot. No significant differences across tratments and genotypes (Two-way ANOVA; data are expressed as mean ± SEM). c Representative western blot of HEK293T cells over-expressing WT, G2019S, or G2019S/K1906R (kinase-dead) LRRK2 probed for total (UDD3) and phosphorylated (pS1292-LRRK2) LRRK2, LIMP1, GCase, phosphorylated Rab29 (T71), and γ-tubulin. LRRK2 kinase function, increased autophosphrylation and phosphorylation of endogenous Rab29 in cells expressing G2019S-LRRK2 is prevented by the kinase inactivating mutation (K1906R). d Representative western blot of cytoplasmic and lysosomal fraction of HEK293T cells over-expressing WT, G2019S, or G2019S/K1906R (kinase-dead) LRRK2 probed for total (UDD3) and phosphorylated (pS1292-LRRK2) LRRK2, LIMP1, GCase, phosphorylated Rab29 (T71), and γ-tubulin. e Quantification of GCase activity normalized by GCase level in the lysosomal fraction of HEK293T cells over-expressing WT, G2019S, or G2019S/K1906R (kinase-dead) LRRK2 showed a significant increase in G20129S samples compared with LRRK2 WT or with the kinase-dead mutant. (One-way ANOVA with Tukey’s post-test; *p < 0.05; data are expressed as mean ± SEM).